MyD88 in myofibroblasts enhances nonalcoholic fatty liver disease-related hepatocarcinogenesis via promoting macrophage M2 polarization

Background Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver diseases and has emerged as the leading factor in the pathogenesis of hepatocellular carcinoma (HCC). MyD88 contributes to the development of HCC. However, the underlying mechanism by which MyD88 in myofibroblasts regulates NAFLD-associated liver cancer development remains unknown. Results Myofibroblast MyD88-deficient (SMAMyD88−/−) mice were protected from diet-induced obesity and developed fewer and smaller liver tumors. MyD88 deficiency in myofibroblasts attenuated macrophage M2 polarization and fat accumulation in HCC tissues. Mechanistically, MyD88 signaling in myofibroblasts enhanced CCL9 secretion, thereby promoting macrophage M2 polarization. This process may depend on the CCR1 receptor and STAT6/ PPARβ pathway. Furthermore, liver tumor growth was attenuated in mice treated with a CCR1 inhibitor. CCLl5 (homologous protein CCL9 in humans) expression was increased in myofibroblasts of HCC and was associated with shorter survival of patients with HCC. Thus, our results indicate that MyD88 in myofibroblasts promotes NAFLD-related HCC progression and may be a promising therapeutic target for HCC treatment. Conclusion This study demonstrates that MyD88 in myofibroblasts can promote nonalcoholic fatty liver disease-related hepatocarcinogenesis by enhancing macrophage M2 polarization, which might provide a potential molecular therapeutic target for HCC. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-024-01489-x.


Introduction
Liver cancer is one of the most common cancers and the third leading cause of cancer-related deaths worldwide [1].Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer, accounting for approximately 90% of primary liver cancers.HCC frequently occurs in patients with underlying chronic liver disease, mainly caused by hepatitis B and hepatitis C virus infections, excessive alcohol consumption, and nonalcoholic fatty liver disease (NAFLD) [2].Recent epidemiological studies have shown that the annual incidence of NAFLD-associated HCC is increasing [3,4].NAFLD encompasses a spectrum of liver pathologies, ranging from simple steatosis (NAFL), nonalcoholic steatohepatitis (NASH) and fibrosis/cirrhosis; these conditions can progress to HCC [5].Thus, it is crucial to obtain a better understanding of the obesity-associated tumor-promoting mechanisms underlying HCC development.
The tumor microenvironment (TME) includes various stromal cells, such as fibroblasts, endothelial cells, adipocytes and immune cells, as well as noncellular components, which are known to play critical roles in tumor progression and therapeutic responses [6,7].Among TME stromal cells, cancer-associated fibroblasts (CAFs) are one of the most abundant cellular components [8,9].Hepatic stellate cells (HSCs) are the main source of CAFs in HCC.Activated HSCs, highly express α-smooth actin (α-SMA), act as fibroblasts, produce ECM proteins, and play a key role in liver fibrosis [10].CAFs contribute to HCC development by reducing immune surveillance, promoting tumor angiogenesis, and secreting regulatory molecules, and particularly, synthesizing and remodeling the ECM [11,12].The progression of NAFLD to HCC is characterized by inflammation and fibrosis.Hepatic injuries eventually lead to HSCs activation, collagen deposition, and ECM deposition, consequently promoting fibrosis and cirrhosis [13,14].However, the mechanism by which HSCs promote NAFLD and lead to HCC development remains poorly understood.
Toll-like receptors (TLRs) are pattern recognition receptors that mediate the recognition of pathogen-associated molecular patterns [15].Myeloid differentiation factor 88 (MyD88), a ligand of TLRs, is a signaling adaptor protein shared by all TLRs, except for TLR3, that activates proinflammatory cascades [16,17].MyD88 deletion increases the risk of developing type 2 diabetes and hepatic steatosis in animals fed with a high-fat diet (HFD) [18,19].Furthermore, hepatocyte-specific deletion of MyD88 is a predisposing factor for glucose intolerance, inflammation, and hepatic insulin resistance (IR) [20].In contrast, deleting MyD88 in intestinal epithelial cells or myeloid cells protects mice from obesity and IR [18,21,22].MyD88 also promotes the development of diethylnitrosamine (DEN)induced hepatocarcinogenesis [23].However, the contributions of MyD88 to NAFLD-related HCC have not yet been reported.MyD88 is widely expressed in various liver cells, including hepatocytes, HSCs, and Kupffer cells.In myofibroblasts, MyD88 inhibits hepatocarcinogenesis by preventing aerobic glycolysis of liver cancer cells in DEN/ CCl 4 -induced HCC [24].However, there is limited information about the function of MyD88 in myofibroblasts in NAFLD and NAFLD-related liver cancer.
In the present study, we explored the HSC-specific role of TLR signaling in NAFLD-related HCC development using mice with a genetic deletion of MyD88 in myofibroblasts.Myofibroblast-specific MyD88 deletion decreased fat accumulation and tumor incidence in HFD-induced NAFLD and DEN/HFD-induced liver cancer.

Mice
MyD88 fl/fl and α-SMA-cre mice on a C57BL/6 background have been described previously [24,25].Mice with a conditional knockout of MyD88 in

Graphical Abstract
α-SMA-expressing myofibroblasts (SMA MyD88−/− ) were generated by crossing MyD88 fl/fl and α-SMA-cre mice.Control mice were cre-negative littermates.All mice were maintained in specific pathogen-free and humidity-and temperature-controlled microisolator cages with a 12-h light/dark cycle at the Institute of Biophysics, Chinese Academy of Sciences.All animal studies were performed after being approved by the Institutional Laboratory Animal Care and Use Committee of the Beijing Jiaotong University.

HFD-induced nonalcoholic fatty liver
Male mice were fed normal diet (ND, 10% of total energy from fat, Huafukang, Beijing, CN) or were fed HFD (60% of total energy from fat, Huafukang, Beijing, CN) beginning at 6 weeks of age to induce obesity for 12 weeks.

DEN/HFD-induced hepatocellular carcinoma
The male mice were i.p. injected with DEN (50 µg/g body weight) (Sigma-Aldrich, St. Louis, MO, USA) at the age of 15 days and were fed HFD (60% of total energy from fat, Huafukang, Beijing, CN) beginning at 6 weeks of age.Liver tumorigenesis was monitored for 10 months.

Mouse transplanted tumor model
Groups of MyD88 fl/fl and SMA MyD88−/− mice were fed with HFD for 3 months.Exponentially growing Hepa1-6 cells were harvested and washed, and a suspension of 1 × 10 6 cells in 200 µl of sterilized phosphate-buffered saline was injected subcutaneously into the abdomen region of MyD88 fl/fl and SMA MyD88−/− mice.The mice were continued fed with HFD.Two days later, the mice received vehicle or 3 mg/kg CCR1 inhibitor J113863 by intraperitoneal injection twice a week for 2 weeks.Tumor growths were measured for 2 weeks.

Blood biochemical assays
Blood samples of mice were centrifuged at 3000 rpm for 8 min to obtain serum.The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) were detected by Beijing Vital River Laboratory Animal Technology (Beijing, China).

Isolation of primary HSCs
Primary HSCs was isolated from livers as previously described using eight-week-old mice.The mice were anesthetized with 3% ethyl carbamate and treated with phosphate balanced solution and 0.16 mg/mL collagenase I for liver perfusion.The liver was removed, divided it into small pieces, and was placed in the digestive buffer at 37 °C for 30 min.The filtered cells were centrifuged at 50×g for 2 min to remove hepatocytes.For HSC enrichment, the remaining NPC fraction was resuspended in 11.5% OptiPrep (Axis-Shield, Oslo, NOR) and placed between a bottom cushion of 15% OptiPrep and a top layer of PBS [27].After centrifugation at 1400×g for 20 min, the HSC fraction at the interface between the top and intermediate layers was obtained.The purity of the HSC fraction was estimated based on the autofluorescence signal.

Cellular immunofluorescence
LX-2 cells were plated into 24-well plates.For fluorescence staining, the cells were fixed in 4% formaldehyde for 15 min and permeabilized with 0.2% Triton-X 100 for 10 min at room temperature.The cells were incubated with 2% BSA to block nonspecific binding sites.Then, the cells were incubated with anti-MyD88, anti-α-SMA antibodies (Abcam, Cambridge, Cambs, UK) and anti-CD206 antibodies (BD Pharmingen, San Diego, CA, USA), followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA).

RNA sequencing analysis
RNA-sequencing analyses were performed in DEN/HFDinduced HCC tissues from control and SMA MyD88−/− mice.Total RNA was extracted with RNeasy Mini Kit (QIAGEN, Dusseldorf, Germany), and RNA-sequencing analyses were performed on the BGISEQ-500 sequencer platform by BGI (Shenzhen, China).Stats package and plots with ggplot2 (RRID:SCR_014601) package in R (version 3.5) were used in principle component analysis.The raw transcriptomic reads were mapped to C57BL/6 genome using HISAT40/Bowtie241 tools after removing adaptor sequences, reads containing polyN sequences, and low-quality reads.Normalization was performed and RESM software was used.Significantly differentially expressed genes (DEGs) were identified by setting padj < 0.05, and the absolute value of log2 Ratio ≤ 0.5.The KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed by using phyper in R. All data mining, and figure presentation were conducted on the Dr Tom network platform of BGI (http:// report.bgi.com).

Western blot analysis
Western blot was performed as previously described.Tissue and cell extracts were separated by electrophoresis on a 10% SDS-PAGE gel at 65 V for 45 min and 115 V for 1 h 20 min, then transferred to a PVDF membrane at 200 mA for 1 h.Membranes were blocked with 5% milk in TBST for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-MyD88, anti-α-SMA (Abcam, Cambridge, Cambs, UK), anti-FASN (Santa Cruz Biotechnology, TX, USA), anti-SREBP1 (Santa Cruz Biotechnology, TX, USA), anti-SCD1 (Santa Cruz Biotechnology, TX, USA), anti-STAT6 and anti-p-STAT6 antibodies (Affinity Biosciences, Liyang, Jiangsu, CN).HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were used as secondary antibodies.Blots were scanned using a Clinx Science Instrument.All specific bands were quantified with the ImageJ (version 1.8 RRID:SCR_003070) automated Digitizing System.

Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was isolated respectively from frozen liver tissues and cells by TRIzol reagent (Invitrogen, USA), and then up to 1 µg of RNA was reverse transcribed by Prime Script RT Master Mix Kit (Tiangen Biotech, Beijing, CN). cDNA was duplicated by SYBR Premix ExTaqTM Kit (Tiangen Biotech, Beijing, CN).The primer sequences are listed in Supplementary Table 1.Data were analyzed using the 2 −ΔΔCt method and normalized to β-actin expression as previously described.

Clinical samples
Primary human HCC and adjacent nontumor liver tissue samples were obtained from HCC patients at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, with informed consent obtained from all patients.The Ethics Committee of Beijing Jiaotong University approved the use of human specimens in accordance with the Declaration of Helsinki.

Public database analysis
Gene expression data (GSE164760, GSE190967 profiling data) were downloaded as raw signals from the Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/ geo), analyzed using the Geo2R function from NCBI (https:// www.ncbi.nlm.nih.gov/ geo/ geo2r).The rank sum test was applied to evaluate the differences in the median CCL15 expression levels between patients with NASH or HCC and healthy controls.Pearson's correlation analysis was performed on α-SMA and CCL15 or α-SMA and CCR1.Gene expression of HCC was obtained from published gene expression profiles included in the TCGA dataset (https:// www.cancer.gov/).Survival analysis was performed with MyD88, α-SMA and CCL15, combined α-SMA/MyD88/CCR1 gene expression bifurcated by the spline method to group into low vs. high.Overall survival was calculated by the Kaplan-Meier method.Differences were analyzed by the log-rank test.

Quantitative and statistical analysis
The data were expressed as means ± SEM and analyzed using GraphPad Prism (version 7.0; RRID:SCR_002798).Differences between the two groups were compared using two-tailed unpaired Student's t-test analysis.Twoway ANOVA was used for multiple comparisons.For all tests, p < 0.05 was considered statistically significant.p < 0.01 was considered extremely significant.

MyD88 expression in myofibroblasts was upregulated during the progression of NAFLD-related HCC
To explore the correlation between MyD88 expression in the mouse liver and development of NAFLD-related HCC, we used C57BL/6 mouse models of NAFLD and HFD-related HCC.We employed a short-term HFD model as an NAFLD model (Fig. 1A), and revealed an increasing aggravation of liver impairment in fatty liver tissues compared with normal liver tissues via H&E staining.Oil red O staining showed increased liver fat accumulation.Immunofluorescence staining indicated that MyD88 expression in α-SMA + cells was significantly upregulated in fatty liver tissues (Fig. 1B).The DEN/ HFD model is an NAFLD-related HCC model (Fig. 1C).Consistent with the fatty liver tissue results, fat accumulation, and the proportion of MyD88 and α-SMA doublepositive cells were increased in HCC tissues (Fig. 1D-E).MyD88 and α-SMA protein levels (Fig. 1F) were consistent with immunofluorescence staining (Fig. 1D).Thus, MyD88 expression in myofibroblasts appears correlated with the pathogenesis of NAFLD-related HCC.

MyD88 deficiency in myofibroblasts attenuated fat accumulation in HFD-induced NAFLD
To investigate the role of MyD88 in myofibroblasts, we conditionally deleted MyD88 in murine myofibroblasts as described previously [28,29].We crossed mice carrying the loxP-flanked MyD88 allele with α-SMA-cre mice to achieve MyD88 ablation specifically in myofibroblasts (SMA MyD88−/− ) mice.The absence of MyD88 in the HSCs derived from SMA MyD88−/− mice was confirmed by double immunofluorescence staining (Fig. 2A) and by reduced MyD88 protein levels in HSCs derived from SMA MyD88−/− mice livers compared with those in HSCs derived from MyD88 fl/fl mice (Fig. 2B).
To explore the role of MyD88 in NAFLD, SMA MyD88−/− mice and control littermates were used to establish an HFD-induced NAFLD model.We found a significant reduction in the body weight gain and liver weight of SMA MyD88−/− mice compared with those of MyD88 fl/ fl mice fed with an HFD (Fig. 2C-E).SMA MyD88−/− mice also showed impaired glucose tolerance compared with MyD88 fl/fl mice (Fig. 2F).Liver fat accumulation is frequently accompanied with elevated ALT, AST, TG and total TC levels.As shown in Fig. 2G-J, SMA MyD88−/− mice exhibited lower serum levels of ALT, AST, TG and TC.H&E and oil red O staining demonstrated that myofibroblast-specific MyD88 deletion impaired fat accumulation (Fig. 2K).Myofibroblast-specific MyD88 deletion also decreased the expansion of myofibroblasts, as detected via α-SMA immunostaining (Fig. 2K).There were no significant differences between SMA MyD88−/− and MyD88 fl/ fl mice fed with a normal diet (Fig. 2K).Compared with MyD88 fl/fl mice, the downregulated protein levels of MyD88 and α-SMA in the liver tissues of SMA MyD88−/− mice were further confirmed by western blot (Fig. 2L-M).In summary, MyD88 knockout in myofibroblasts significantly attenuated liver injury, and fat accumulation in HFD-induced NAFLD.

MyD88 deficiency in myofibroblasts attenuated DEN/ HFD-induced NAFLD-related hepatocarcinogenesis
To explore the role of MyD88 in NAFLD-related liver cancer, SMA MyD88−/− and MyD88 fl/fl mice were first treated with an intraperitoneal injection of 50 µg/g DEN (Sigma-Aldrich, St. Louis, MO, USA) at the age of 15 days and 30 days, and then fed with an HFD beginning at 6 weeks of age.Liver tumorigenesis was monitored for 10 months.SMA MyD88−/− mice showed significantly reduced body weight gain (Fig. 4A) All control mice developed liver tumors within 30 weeks; however, SMA MyD88−/− mice showed obvious resistance to liver cancer development; approximately 77.78% of SMA MyD88−/− mice developed tumors (Fig. 4B-C).Myofibroblast-specific MyD88 deletion significantly decreased liver weight, liver tumor size at 10 months and number of liver tumors (Fig. 4D-F).SMA MyD88−/− mice also demonstrated impaired glucose tolerance (Fig. 4G).Compared with control mice, the serum TG and TC levels were significantly decreased in DEN/HFD-treated SMA MyD88−/− mice (Fig. 4H-I).H&E staining, immunohistochemical staining of Ki67, Sirius red staining and Oil red O staining also revealed that DEN/HFD-treated SMA MyD88−/− mice had reduced inflammatory cell infiltration, steatosis, cell proliferation, fibrosis and fat accumulation compared with MyD88 fl/fl mice (Fig. 4J).Immunofluorescence staining and western blot indicated that MyD88 and α-SMA expression was significantly downregulated in the liver of SMA MyD88−/− mice compared with that in the liver of DEN/HFD-treated MyD88 fl/fl mice (Fig. 4J-K).Thus, MyD88 knockout in myofibroblasts significantly alleviates NAFLD-related hepatocarcinogenesis.

MyD88 deficiency in myofibroblasts attenuated macrophage M2 polarization and fat accumulation in NAFLD-related HCC
Consistent with NAFLD results, MyD88 deletion in myofibroblasts significantly decreased the infiltration of F4/80 + macrophages, Gr1 + neutrophils, and CD11b + monocytes in NAFLD-related HCC (Fig. 5A-B).HCC is characterized by considerable macrophage infiltration, raising questions about the classification of infiltrating macrophages in liver cancer.We therefore analyzed the number of CD206 + (an M2 marker) and CD86 + (an M1 marker) macrophages in HCC tissues and revealed that SMA MyD88−/− mice had enhanced M1-like phenotype and reduced M2-like phenotype compared with control mice (Fig. 5A-B).
We further examined lipogenesis gene expression in HCC tissues.As shown in Fig. 5E, SREBP1-c and SCD1 mRNA levels were markedly downregulated in HCC tissues derived from SMA MyD88−/− mice.Compared with MyD88 fl/fl mice, the protein expression levels of FASN, SREBP1, and SCD1 were consistently downregulated in HCC tissues of SMA MyD88−/− mice (Fig. 5F).In summary, MyD88 deletion in myofibroblasts significantly attenuated macrophage M2 polarization and fat accumulation in NAFLD-related HCC.

Specific deletion of MyD88 in myofibroblasts reduced CCL9 secretion in NAFLD-related HCC
To further identify the gene expression signature, we performed a protein-coding mRNA-seq analysis of HCC tissues derived from DEN/HFD-treated SMA MyD88−/− mice and control mice.A total of 344 differentially expressed genes (DEGs) were identified, including 58 upregulated and 286 downregulated genes (Fig. 6A; padj < 0.05; absolute value of log2 ratio ≥ 1).
A subsequent pathway analysis showed that MyD88 deletion in myofibroblasts mainly promoted the changes in metabolism related pathways, including the metabolism of lipids, amino acids and sugars (Fig. 6B).The expression of genes related to lipid metabolism: Ctps2, Mvd, Acsl4, Pck2, and Pgm2 was significantly downregulated (Fig. 6C-D).Among the 30 genes with the most significant differences in expression, the expression of CCL9, which encodes an inflammatory chemokine, was significantly decreased in DEN/HFDtreated SMA MyD88−/− mice compared with controls (Fig. 6E).Double immunofluorescence staining showed that CCL9 was highly expressed in SMA + cells in the HCC tissues of control mice, indicating that CCL9 was expressed in myofibroblasts.Consistent with the mRNAseq results, the number of CCL9 + cells in the HCC tissues of SMA MyD88−/− mice was significantly decreased (Fig. 6F).CCL9 protein expression levels were further analyzed via ELISA using LX-2-conditioned media (CM).CCL15 (homologous protein CCL9 in humans) secretion, was consistently increased in LX-2 cells induced by HepG2 conditioned media (TCM) and OA; meanwhile, the MyD88 inhibitor ST2825 greatly suppressed TCM and OA-induced CCL15 protein levels in LX-2 cells (Fig. 6G).Thus, myofibroblast-derived CCL9 may play an important role in NAFLD-related HCC.

CCL9 promoted M2 polarization and fat accumulation in macrophages by activating the STAT6/ PPARβ pathway in HCC
We analyzed the HSC-specific role of MyD88 in macrophage polarization in vitro.As MyD88 deletion in myofibroblasts decreased CCL9 secretion, we hypothesized that MyD88 in myofibroblasts promotes the polarization of macrophages through CCL9.RAW264.7 cells were cultured with IL-4 and IL-13 for 48 h to induce M2 polarization.Compared with untreated DMEM group, treatment with IL-4 and IL-13 activated CD206 expression in macrophages.Immunostaining results indicated that OA and CCL9 recombinant proteins further upregulated CD206 expression in macrophages.However, treatment of RAW264.7 cells with J113863 (a CCL9 receptor CCR1 inhibitor) eliminated CCL9-induced expression of CD206 (Fig. 7A).After M2 polarization induction and OA treatment, IL-10, Arg1, and YM1 levels in RAW264.7 cells were significantly upregulated compared to controls.Exogenous addition of CCL9 recombinant protein further promoted M2-related gene expression (Fig. 7B).
Signal transducer and activator of transcription 6 (STAT6) and PPARβ are known to drive macrophage M2 polarization.Higher levels of STAT6 and PPARβ are found in M2 macrophages [30].To determine how myofibroblast-derived MyD88 affects macrophage polarization, we analyzed HCC tissue and macrophages using western blot.The results revealed that SMA MyD88−/− mice had lower levels of phosphorylated STAT6, PPARβ and Arg1 than MyD88 fl/fl mice (Fig. 7C).Consistent with animal experiment results, when IL-4 and IL-13 induced M2 macrophages were cultured with OA, the exogenous CCL9 recombinant protein significantly upregulated p-STAT6 and Arg1 expression (Fig. 7C).In addition, M2 macrophages treated with J113863 simultaneously  demonstrated decreased p-STAT6 levels and PPARβ expression (Fig. 7D).These results suggest that CCL9 promotes M2-type polarization of macrophages via the STAT6/PPARβ signaling pathway.

Blocking of CCL9 inhibited liver tumor growth by preventing M2 polarization of macrophages in the transplanted tumor model
To further explore the role of CCL9 in liver cancer progression, MyD88 fl/fl and SMA MyD88−/− mice were used to establish the subcutaneous transplantation model for Hepa1-6 cells.The mice were fed with HFD for 3 months, after which 1 × 10 6 Hepa1-6 cells were subcutaneously in these mice.The mice were fed with HFD and blocked by intraperitoneal injection of CCL9 receptor CCR1 inhibitor J113863, Tumor growth was evaluated for 2 weeks.MyD88 knockout in myofibroblasts significantly inhibited tumor growth.Similar effects were observed in the CCR1 inhibition group (Fig. 7E-F).When M2 polarization was analyzed using CD206 immunofluorescence staining, we found that SMA MyD88−/− and J113863-injected MyD88 fl/fl mice had attenuated M2-type polarization compared with MyD88 fl/fl mice.Furthermore, SMA MyD88−/− mice, showed decreased CCL9 expression compared with MyD88 fl/fl mice as determined via CCL9 immunofluorescence staining (Fig. 7G).In addition, the numbers of CCL9 + /MyD88 + cells were significantly decreased in SMA MyD88−/− and J113863-injected MyD88 fl/fl mice (Fig. 7G-H).We revealed that, MyD88 in myofibroblasts promotes M2 polarization via CCL9 secretion in transplanted tumors.Simultaneously, inhibited the CCL9 receptor CCR1 to block CCL9, exerting an antitumor effect.

CCLl5 expression was increased in myofibroblasts of HCC and was associated with shorter survival of patients with HCC
To extend our findings to human samples, we performed immunofluorescence double staining of α-SMA and CCL15 in tumor tissues and adjacent paracancerous tissues derived from patients with HCC.Compared with paracancerous tissues, the number of α-SMA + fibroblasts expressing CCL15 evidently increased in tumor tissues (Fig. 8A).Furthermore, we analyzed the publicly available GEO dataset GSE164760, and compared the CCL15 expression of healthy, NASH and HCC tissues.Compared with healthy controls, CCL15 mRNA levels were significantly increased in NASH and HCC tissues.CCL15 expression level in tumor tissues was also higher than that in HCC paracancerous tissues.Next, we found significant positive correlations among CCL15, CCR1 and α-SMA in HCC by correlation analysis with GEO data set GSE164760 and GSE190967 (Fig. 8C-E).To further support the α-SMA-MyD88-CCL15 axis in HCC, we performed survival analysis using RNA-seq data acquired from the TCGA database (http:// cance rgeno me.nih.gov/).We found that the high level of α-SMA, MyD88 or CCL15 significantly impaired the overall survival compared with their corresponding low levels (Fig. 8F-H).In addition, a combined high expression level of α-SMA-MyD88-CCR1 significantly impaired overall survival compared with their low expression level (Fig. 8I).Altogether, these results suggest that the upregulation of MyD88 and CCL15 in myofibroblasts is a poor prognostic factor of HCC.

Discussion
In this study, we explored the role of the MyD88 signaling pathway in HSCs/myofibroblasts during the development from NAFLD to HCC.MyD88 deficiency in myofibroblasts attenuated fat accumulation in HFD-induced NAFLD and NAFLD-related HCC.This study showed that MyD88 in myofibroblasts promotes CCL9/CCL15 secretion, which enhanced macrophage M2 polarization by activating the STAT6/PPARβ pathway to promote hepatocarcinogenesis (Fig. 8J).
Toll-like receptor signaling through MyD88 contributed to hepatocarcinogenesis. MyD88 deficient mice developed fewer and smaller HCC tumors than WT mice in DENinduced hepatocarcinogenesis [23].In hepatocytes, MyD88 signaling promoted HBV-mediated liver carcinogenesis [32].In Kupffer cells, resulted in secretion of IL-6 which contributed development of DEN-induced hepatocarcinogenesis [23].Recently, we also found that MyD88 in HSCs/myofibroblasts promoted the progress of DEN/CCl 4 -induced fibrosis-related HCC via CCL20 secretion and promoted the aerobic glycolysis of liver cancer cells [28].Although MyD88 is widely expressed by different cells, the cell typespecific role of MyD88 in NAFLD-related HCC is mostly unknown.Antje Mohs et al. investigated the role of MyD88 during progression from NASH to HCC using a mouse model of chronic liver injury model.They found that MyD88 deficiency impaired HCC formation; however, hepatocyte specific MyD88 deletion did not affect disease progression.These results suggested that MyD88 in non-parenchymal liver cells was necessary for carcinogenesis during chronic liver injury [33].The current study revealed that the specific deletion of MyD88 in HSCs/myofibroblasts attenuated DEN/HFD-induced hepatocarcinogenesis (Fig. 4), hepatic steatosis, cell proliferation, and lipogenesis (Fig. 5).These findings were consistent with those of the NAFLD model.This is the first report to describe the myofibroblast-specific role of MyD88 signaling in NAFLD-related HCC.Myofibroblast-specific MyD88 promotes NAFLD-related HCC.
CAFs are the most important stromal components in HCC and are crucial for HCC development and progression, since more than 80% of HCCs develop in fibrotic or cirrhotic livers [11,34].CAFs in HCC are mostly derived from HSCs, and α-SMA is a commonly used marker for CAFs [35].Activated HSCs promote HCC progression by secreting cytokines such as HGF and CCL20 [28,36] and crosstalk with hepatocytes, macrophages, and other stromal cells [28,34,37].The progression of NAFLD to NASH and liver fibrosis is closely associated with a series of liver injury resulting from lipotoxicity, oxidative stress, or ER stress.These injuries activate HSCs, enhance collagen deposition, and promote liver fibrosis and cirrhosis [38,39].In addition, altered ECM composition in fibrotic or cirrhotic NASH livers contributes to carcinogenesis [40,41].However, the detailed mechanism of HSC in NAFLD-related HCC has not been elucidated.We found that MyD88 in HSCs promotes macrophage M2 polarization and enhances the progression of NAFLD to HCC by secreting CCL9 (Fig. 8J).
TAMs are the most abundant immune cells in the TME and exert import anti-tumor or pro-tumor effects depending on the balance of macrophage M1/M2 polarization [42,43].In HCC, TAMs promote tumor growth, angiogenesis, invasion, and metastasis by releasing cytokines, chemokines, and matrix metalloproteinases (MMPs).In HCC, crosstalk between activated HSCs and macrophages showed that interaction with CD68 and regulation of GAS6 expression by endosialin in fibroblasts drives recruitment and polarization of macrophages in HCC [37].
In conclusion, MyD88 plays a promoting role in myofibroblasts in NAFLD and NAFLD-related HCC.MyD88 in myofibroblasts promotes fat accumulation by enhancing lipid droplet accumulation in HSCs and promotes macrophage M2 polarization and enhances NAFLD to HCC progression by secreting CCL9/CCL15.MyD88 in HSCs/myofibroblasts may be a potential therapeutic and/ or preventative target for NAFLD and HCC.

Fig. 1
Fig. 1 MyD88 expression in myofibroblasts was associated with NAFLD-related liver cancer.C57BL/6 mice were used for NAFLD model ( n = 5 per group) and liver cancer model ( n = 10 per group).A Schematic representation of the HFD induced NAFLD model.B Representative staining of H&E, Oil red O, and double staining of α-SMA and MyD88 in normal liver and NAFLD samples.Scale bar, 50 μm.C Schematic representation of the DEN/HFD induced liver cancer model.D Representative staining of H&E, Oil red O, and double staining of α-SMA and MyD88 in normal and HCC samples.Scale bar, 50 μm.E Statistical analysis (magnification, 200×).*** p < 0.001.F The expression levels of MyD88 in liver tissues were determined by western blot.The densities of proteins were quantified using densitometry.Proteins were normalized to β-actin.*p<0.05,**p<0.01.HFD: High fat diet; NAFLD: Non-alcoholic fatty liver disease; DEN: N-nitrosodiethylamine

Fig. 2
Fig. 2 MyD88 deficiency in myofibroblasts attenuated fat accumulation in HFD-induced NAFLD.MyD88 fl/fl and SMA MyD88−/− mice were fed with HFD to establish a NAFLD model (n = 5 per group), and the control group was fed with normal diet.The data are representative of at least three independent experiments.A Primary hepatic stellate cells were isolated from MyD88 fl/fl and SMA MyD88−/− mice liver, immunofluorescence double staining of α-SMA (red) and MyD88 (green) in liver tissues (Scale bar, 50 μm).B The protein level of MyD88 in the primary hepatic stellate cells of MyD88 fl/fl and SMA MyD88−/− mice were measured using western blot.C Body weight at end of diet.D Representative images of the livers from SMA MyD88−/− mice and control mice.E Liver weight, (F) Glucose tolerance test (GTT), (G) ALT, (H) AST, (I) TG and (J) TC levels of MyD88 fl/fl and SMA MyD88−/− mice are shown.* p < 0.05, ** p < 0.01.*** p < 0.001.K Representative H&E, Oil red O staining and staining of α-SMA in liver tissues of MyD88 fl/fl and SMA MyD88−/− mice.Scale bar, 50 μm.Statistical analysis.** p < 0.01 (L-M) The protein levels of MyD88 and α-SMA in liver tissues of MyD88 fl/fl and SMA MyD88−/− mice were determined by western blot.The densities of proteins were quantified using densitometry.Proteins were normalized to β-actin.* p < 0.05, ** p < 0.01.ALT: alanine aminotransferase; AST: aspartate aminotransferase; TG: triglyceride; TC: total cholesterol

Fig. 3
Fig. 3 MyD88 deficiency in myofibroblasts attenuated liver inflammation and lipid metabolism-associated gene expression in HFD-induced NAFLD.A-D MyD88 fl/fl and SMA MyD88−/− mice were used for NAFLD model (n = 5 per group), and the control group was fed with normal diet.The data are representative of at least three independent experiments.A Representative staining of F4/80, Gr1 and CD11b in liver tissues (Scale bar, 50 μm) and (B) Statistical analysis (magnification, 200×).*** p < 0.001.C The mRNA levels of FANS2, ACC1 and SREBP1-c in the liver tissues of MyD88 fl/fl and SMA MyD88−/− mice were measured using qPCR analysis.** p < 0.01, *** p < 0.001.D The protein levels of FASN, SREBP1 and SCD1 in liver tissue of MyD88 fl/fl and SMA MyD88−/− mice were determined by western blot.The densities of proteins were quantified using densitometry.Proteins were normalized to β-actin.** p < 0.01, *** p < 0.001

Fig. 4
Fig. 4 MyD88 deficiency in myofibroblasts attenuated DEN/HFD-induced liver cancer.Groups of MyD88 fl/fl and SMA MyD88−/− mice were used for the DEN/HFD-induced liver cancer model (n = 9 per group).The data are representative of at least three independent experiments.A Body weight, (B) Representative images of mouse livers, (C) Tumor incidence, (D) Liver weight, E Size of the maximal tumors and (F) Number of tumors per mouse are shown.* p < 0.05, ** p < 0.01.G Glucose tolerance test, serum (H) TG and (I) TC of MyD88 fl/fl and SMA MyD88−/− mice are shown.* p < 0.05, ** p < 0.01.J H&E, Ki67, Sirius Red and Oil red O staining and immunofluorescence staining of α-SMA and MyD88 in liver tissues of MyD88 fl/fl and SMA MyD88−/− mice.Scale bar, 50 μm.K The protein levels of MyD88 and α-SMA in liver tissues of MyD88 fl/fl and SMA MyD88−/− mice treated with DEN/HFD were determined by western blot.The densities of proteins were quantified using densitometry.Proteins were normalized to β-actin.*p<0.05,**p<0.01

Fig. 6
Fig. 6 Secreted CCL9 was critical for DEN/HFD induced hepatocarcinogenesis. RNA sequencing analysis of DEGs between DEN/HFD-induced liver cancer tissues from MyD88 fl/fl and SMA MyD88−/− mice.A Volcano diagram of DEGs, the threshold is padj < 0.05 and the absolute value of log2 Ratio ≥ 1. B KEGG enriched signal pathway analysis with down-regulated genes in liver tissues.C Heatmap view of the most significant differential expressed genes.D Fat accumulation related gene expression in liver tissues.E Gene expression in liver tissues.F Immunofluorescence double staining for CCL9 and α-SMA in liver cancer tissues from MyD88 fl/fl and SMA MyD88−/− mice.Scale bar.50 μm.*p<0.05.G The protein levels of CCL15 in LX-2 medium were measured using ELISA.*p<0.05